Diallyl disulfide (DADS) has shown potential as a therapeutic agent in various cancers. Previously, we found that myeloid cell leukemia sequence 1 (Mcl1) was downregulated in DADS-induced cell cycle arrest in HL-60 human leukemia cells. Here, we investigated the role of this protein in DADS-induced G2/M cell cycle arrest in HL-60 cells. We demonstrated that DADS treatment significantly increased the proportion of G2/M phase HL-60 cells (P<0.05) and caused a time-dependent significant downregulation of Mcl1 and the cell cycle-related proteins PCNA and CDK1 (P<0.05). Small interfering RNA-mediated knockdown of Mcl1 expression in HL-60 cells arrested the cell cycle in G2/M phase. By co-immunoprecipitation, we demonstrated that Mcl1 associated with PCNA and CDK1 in G2/M cell cycle arrest in DADS-treated HL-60 cells. DADS decreased the interaction of Mcl1 with PCNA and CDK1, leading to G2/M cell cycle arrest in HL-60 cells. Mcl1 plays an important role in DADS-induced G2/M cell cycle arrest in HL-60 human leukemia cells.